A multistep process involving adhesive and signaling events regulates inflammatory responses to infection or tissue injury. During inflammation leukocytes adhere to the vascular endothelium and enter subendothelial tissue, an interaction which is mediated by specific binding of the selectin or LEC-CAM class of proteins to ligands on target cells (Lowe, J B et al. 1997 J. Clin. Invest. 99:822-826). Such selectin-mediated cellular adhesion also occurs in thrombotic disorders and parasitic diseases and may be implicated in metastatic spread of tumor cells (Lowe, J B et al. 1997 J. Clin. Invest. 99:822-826).
The selectin proteins are characterized by a N-terminal lectin-like domain, an epidermal growth factor-like domain, and regions of homology to complement binding proteins (Bevilacqua, M P et al. 1993 J. Clin. Invest. 91:379-387). Thus far three human selectin proteins have been identified, E-selectin (formerly ELAM-1), L-selectin (formerly LAM-1) and P-selectin (formerly PADGEM or GMP-140). E-selectin is induced on endothelial cells several hours after activation by cytokines, mediating the calcium-dependent interaction between neutrophils and the endothelium. L-selectin is the lymphocyte homing receptor, and P-selectin rapidly appears on the cell surface of platelets when they are activated, mediating calcium-dependent adhesion of neutrophils or monocytes to platelets. P-selectin is also found in the Weibel-Palade bodies of endothelial cells; upon its release from these vesicles P-selectin mediates early binding of neutrophils to histamine- or thrombin-stimulated endothelium.
Selectins are believed to mediate adhesion through specific interactions with ligands present on the surface of target cells (Varki, A. J. 1997 J. Clin. Invest. 99:158-162). Generally the ligands of selectins are comprised at least in part of a carbohydrate moiety (e.g., sialyl Lewisx and sialyl Lewisa, heparan sulfate) and each selectin appears to bind to a range of carbohydrates with varying affinities. The strength of the selectin mediated adhesive event (binding affinity) may also depend on the density of the carbohydrate and on the density of the selectin on the cell surface. For example, P-selectin binds to carbohydrates containing the non-sialated form of the Lewisx blood group antigen and with higher affinity to sialyl Lewisx. P-selectin may also recognize sulfatides, which are heterogeneous 3-sulfated galactosyl ceramides, isolated from myeloid and tumor cells by lipid-extraction. In addition, the PSGL-1 glycoprotein acts as a ligand for P-selectin on human endothelial cells and platelets (U.S. Pat. No. 5,827,817).
The initial tethering of leukocytes to activated platelets or endothelium is in part mediated by PSGL-1/CD162. PSGL-1 is a mucin-like glycoprotein, expressed on the surface of most cells of hematopoietic origin, which functions as a cellular adhesion molecule. It normally exists as a disulfide linked homodimer with an apparent molecular weight of approximately 240 kD. PSGL-1 is a unique high affinity ligand for P-selectin and is one of several known lower affinity ligands for E-selectin and L-selectin. PSGL-1 has an extracellular domain rich in serines, theronines, and prolines, and which includes 15 decameric repeats, as well as putative sites for tyrosine sulfation (McEver, R P et al. 1997 J. Clin. Invest. 100:485-491). Binding of PSGL-1 to P-selectin requires that PSGL-1 be modified with alpha 2,3-linked sialic acid and alpha 1,3-linked fusose. In addition, PSGL-1 contains a single extracellular cysteine residue located at the junction of the transmembrane domain, followed by a cytoplasmic domain containing putative phosphorylation sites on tyrosine, threonine and serine residues. Comparison of the human and murine PSGL-1 proteins indicates that the transmembrane and cytoplasmic domains are highly conserved, implying an important function for these domains.
Various reports provide evidence that ligation of PSGL-1 on the surface of different cell types, by a variety of agents, results in an intracellular signaling event, including the phosphorylation of multiple intracellular proteins. Moreover, the binding of P-selectin to PSGL-1 may generate signals that are integrated with signals from other mediators to elicit an effector response. For example, the exposure of human monocytes in vitro to immobilized P-selectin has been reported to increase their response to the chemokine RANTES (Hidari, KI-P et al. 1997 J. Biol. Chem. 272:28750-28756). In polymorphonuclear leukocytes (PMNs), PSGL-1 mediated signaling events include activation of MAP kinases and the Ras GTPase, Interleukin-8 (IL-8) secretion, and the rapid tyrosine phosphorylation of a 110 kD protein also associated with the activation of the β2-integrin CD11b/CD18 (Evangelista, V et al. 1999 Blood 93:876-885; Hidari, KI-P et al. 1997 J. Biol. Chem. 272:28750-28756). In addition, adhesion of T lymphocytes to P-selectin has been reported to induce tyrosine phosphorylation of several intracellular proteins including the focal adhesion kinase, pp125FAK (McEver, R P et al. 1997 J. Clin. Invest. 100:485-491). A patient having a variant of leukocyte adhesion deficiency type 1 (LAD-1 variant) syndrome, which causes defects in leukocyte adhesion mediated by B1 and B2 integrins, has been reported. The neutrophils from this patient have a 77% increase in the surface expression of PSGL-1. The mechanism and genetic basis of this individuals' abnormality have not yet been characterized (Harris, E. S. et al. (2001) Blood 97:767-776).
Furthermore, upon leukocyte activation, PSGL-1 can redistribute to the uropods of polarized cells, and may thus interact with cytoskeletal elements during cellular redistribution (McEver, R P et al. 1997 J. Clin. Invest. 100:485-491). The adherence of hematopoietic progenitor cells (HPCs) bearing PSGL-1 on their surface has also been reported to inhibit cell growth and induce apoptosis under certain conditions.
To date, however, there has been no identification of the proximal molecule(s) that interact with the cytoplasmic domain of PSGL-1 and potentially mediate the intracellular signal transduction subsequent to the extracellular ligation of PSGL-1 and/or the cytoskeletal association of PSGL-1.